Maintaining the proper balance between procoagulant and anticoagulant activity within blood vessels is essential for normal hemostasis (Davie, E. W. et al. (1991) Biochemistry, 30(43):10363-10370). Perturbing the balance toward coagulation leads to thrombosis, which can cause heart attack, stroke, pulmonary embolism, and venous thrombosis. There is a need for more effective and safer anticoagulants for the treatment of specific thrombotic disorders.
Tissue factor (“TF”) is a transmembrane glycoprotein that is the major initiator of the coagulation cascade (Nemerson, Y. (1995) Thromb. Haemost. 74(1):180-184). Under normal physiological conditions active TF is not in contact with blood. During vascular injury, exposure to blood of subendothelial TF and collagen leads to activation of coagulation factors and platelets and subsequently to hemostatic plug formation. Exposed TF acts as a cofactor for the factor VIIa (“FVIIa”) catalyzed activation of factor IX (“FIX”) and factor X (“FX”), critical components of the intrinsic tenase and prothrombinase complexes, respectively. This leads to rapid formation of FXa and thrombin. Thrombin then cleaves fibrinogen to fibrin, which subsequently polymerizes to form the fibrin clot. The inappropriate induction of TF expression in a variety of clinical settings can lead to life threatening thrombosis and/or contribute to pathological complications. TF exposure following plaque rupture is believed to be responsible for thrombotic occlusion leading to acute myocardial infarction and stroke. In these settings, proinflammatory signaling pathways activated by coagulation factors also contribute to edema formation and increased infarct size. Vascular injury associated with angioplasty leads to upregulation of TF on SMC's which is believed to induce cell signaling pathways associated with restenosis. TF overexpression in cancer and gram-negative sepsis leads to life threatening thrombosis and activation of inflammatory pathways.
The FVIIa/TF complex is involved in the pathogenic mechanism in a variety of thrombotic diseases and the circulating level of TF is a risk factor for certain patients. FVIIa and TF play unique roles in vascular injury in maintaining hemostasis and initiating thrombosis. TF is expressed in the adventitia normally, but is upregulated and expressed inappropriately in the media and neointima in vascular disease. TF expression in atherosclerotic plaques is increased and shielded from the blood by a thin fibrous cap that may rupture to expose TF. Surgical interventions such as balloon angioplasty, stenting, or endarterectomy damage the vessel wall and expose underlying TF. In the atherosclerotic, lipid-rich, thin-walled plaque, spontaneous rupture or endothelial erosion leads to TF exposure and thrombosis, resulting in unstable angina and myocardial infarction. TF can circulate in cell-derived microparticles and circulating TF levels are elevated in unstable angina, suggesting that this circulating TF may contribute to thrombus formation (Soejima, H. et al. (1999) Circulation 99(22):2908-2913). Often cancer is associated with a hypercoagulable state attributed to overexpression of TF on tumor cells. This predisposes the patient to deep vein thrombosis, pulmonary embolism and low grade disseminated intravascular coagulation (“DIC”). DIC results in microvascular fibrin deposition contributing to multi-organ failure.
Protein based anticoagulants that target TF include TF neutralizing antibodies, active site inhibited factor VIIa (“FVIIai”), tissue factor pathway inhibitor (“TFPI”), and Nematode anticoagulant protein (“NAPC2”). Results from acute arterial injury models of thrombosis indicate that protein based inhibitors of FVIIa/TF are effective antithrombotics, with less bleeding compared to heparin, direct thrombin inhibitors, platelet inhibitors, and FXa inhibitors (Himber, J. et al. (2001) Thromb. Haemost. 85:475-481; Harker, L. A. et al. (1995) Thromb. Haemost. 74(1):464-472. In addition, FVIIa/TF inhibition is superior to other anticoagulants (e.g., heparin, FXa inhibitors) in preventing neointimal thickening and vascular stenosis following balloon injury (Jang, Y. et al. (1995) Circulation 92(10):3041-3050).
Inhibition of TF, FVIIa or the FVIIa/TF complex is an efficacious antithrombotic approach for preventing DIC and reducing mortality in experimental models of sepsis. TFPI analogs prevent both thromboplastin and endotoxin-induced DIC in rabbits (Day, K. C. et al. (1990) Blood 76:1538-1545; Bregengard, C. et al. (1993) Blood Coagul. Fibrinolysis 4:699-706). Monoclonal antibodies against FVIIa (Biemond, B. J. et al. (1995) Thromb. Haemost. 73:223-230) or (Levi, M. et al. (1994) J. Clin. Invest. 93:114-120) prevent endotoxin-induced DIC in monkeys. TF neutralizing antibodies, FVIIai, and TFPI, inhibit DIC and reduce mortality in a baboon model of E. coli-induced sepsis (Creasey, A. A. et al. (1993) J. Clin. Invest 91:2850-2860; Taylor, F. B. et al. (1991a) Blood 78:364-368; Taylor, F. B. et al. (1991b) Circ. Shock 33:127-134; Taylor, F. B. (1996) Haemostasis Suppl. 1 26:83-91). Both free FXa and the FVIIa/TF/FXa complex are known to induce the production of proinflammatory cytokines that are associated with an increased risk of death in patients with sepsis (Riewald, M. et al. (2001) Proc. Natl. Acad. Sci. USA 98:7742-7747). Interestingly, FVIIai was shown to lower plasma levels of IL-6 and IL-8 in the baboon model (Taylor, F. B. et al. (1998) Blood 91:1609-1615), suggesting that FVIIa/TF inhibition may have additional antiinflammatory effects not shared by other anticoagulant mechanisms.
Several antibodies that are effective anticoagulants, which bind to and neutralize either TF or the FVIIa/TF complex or both, have been described (see e.g., Carson, S. D. et al. (1985) Blood 66(1):152-156; Tanaka, H. et al. (1985) Thromb. Res. 40(6):745-756; Kirchhofer, D. et al. (2000) Throomb. Haemost. 84(6);1072-1081; Kirchhofer, D. et al. (2001) Biochemistry 40(3):675-682; Faelber, K. et al. (2001) J. Mol. Biol. 313:83-97; and U.S. Pat. Nos. 5,506,134, 5,986,065, and 6,274,142). The TF targeted antibodies of the present invention are effective anticoagulants that have improved characteristics over previously described TF antibodies. In particular, the antibodies of the invention bind with greater affinity to the FVIIa/TF complex than to TF alone, and are non-competitive with FVII or FX for binding to TF.